In the past several years, the development of techniques capable of detecting DNA sequence variation has aroused a great deal of interest. Major applications include the study of genetic disease, cancer, and identity testing. While DNA sequencing is clearly the most sensitive and informative method, it is too cumbersome for routine use in searching for mutations, especially when the DNA segment of interest is large.
For detection of single-base mutations, alternatives to sequencing consist either of techniques dependent on prior knowledge of the exact base alterations sought, for instance techniques involving allele-specific hybridization (Reinach F. C. et al. Nature (1986) 322:80; Saiki, R. K. et al. Proc Natl Acad Sci USA (1989) 86:6230) or shotgun strategies that ideally would detect any change, for example, SSCP (Orita, M. et al. Genomics (1989) 5:874 and DGGE (Sheffield, B. C. et al. Proc Natl Acad Sci USA (1989) 86:232). Techniques relying on prior knowledge of the base alterations become awkward when sequence differences caused by a variable number of mutations are studied (Stoneking, M. et al. Am J Hum Genet (1991) 48:370). Shotgun strategies can only be applied to small DNA fragments and do not identify the number of modifications or their sites.
The present invention provides a new technique, called low stringency single-specific primer PCR (LSSP-PCR) that, when applied to gene-size DNA fragments, at least up to 1 kb, translates the underlying DNA sequence into a unique multiband "gene signature". Changes as small as single-base mutations significantly alter the signature that is diagnostic of the specific alteration.
Other techniques involving low stringency or random priming have also been disclosed. Dias Neto, E. et al. Nucleic Acids Res (1993) 21:763-764, describe a technique for sex determination by low stringency PCR. This technique utilizes conventional 2-primer PCR but with performance of the reaction under low stringency. When conducted in this way, the reaction mixture yields a pattern rather than a single amplified band. Low stringency conditions are also employed in the performance of random amplified polymorphic DNA (RAPD) as described by Welsch, J. et al. Nucleic Acids Res (1990) 18:7213-7218.